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2153RESEARCH ARTICLE
INTRODUCTIONHair follicle (HF) formation begins with local thickening of the
epithelium to form a placode, and an associated condensation of the
underlying mesenchymal cells termed the dermal condensation
(DC); both structures are common to other ectodermal appendages,
including feathers, teeth and mammary glands. Individual HF
morphogenesis is a tightly regulated process, relying on many highly
conserved signalling pathways, including Delta/Notch,
Wnt/Frizzled, Hedgehog/Patched, TGFβ/BMP and FGF signalling
(Millar, 2002; Schmidt-Ullrich and Paus, 2005), providing a balance
of stimulatory and inhibitory influences. Knockout and transgenic
animal models have revealed that active Wnt signalling is crucial for
the initiation of follicular morphogenesis (Andl et al., 2002;
Huelsken et al., 2001; van Genderen et al., 1994; Zhang et al., 2008),
whereas studies in mice lacking sonic hedgehog (SHH) indicate that
SHH is required after HF initiation, downgrowth of the follicle
epithelium and dermal papilla (DP) formation (Chiang et al., 1999;
Karlsson et al., 1999; St-Jacques et al., 1998). In addition to
individual follicle morphogenesis, the processes that determine
whether surface epithelial cells become interfollicular skin
epidermis or HFs also establish the spatial distribution of these
appendages. In this context, ectodysplasin A (EDA) and its receptor
EDAR appear to play a crucial role (Headon et al., 2001; Headon
and Overbeek, 1999; Laurikkala et al., 2002). EDA and EDAR
interact with members of the bone morphogenetic protein (BMP)
family, some of which are inhibitory to follicle development, to
establish follicle patterning (Mou et al., 2006; Pummila et al., 2007).
In the earliest stages of follicle initiation, there has been an emphasis
on determining the molecular factors that distinguish the placode
versus the interfollicular epidermis (Nowak et al., 2008; Rhee et al.,
2006). Two pathways that have an essential role in these cell fate
decisions include EGF and KGF (FGF7 – Mouse Genome
Informatics) (Beer et al., 2000; du Cros, 1993; Peus and Pittelkow,
1996; Schneider et al., 2008, Guo et al., 1996). Disruption or
blocking of the EGF receptor (EGFR) in several mouse models
results in abnormalities occurring late on in follicle development and
during the adult hair follicle cycle, leading to the hypothesis that
EGF signalling has an important positive influence on follicle
development and growth (Miettinen et al., 1995; Murillas et al.,
1995). EGFR also has several other activating ligands, including
transforming growth factor-α (TGFα), heparin-binding EGF-like
growth factor (HBEGF), amphiregulin (AR, AREG – Mouse
Genome Informatics), betacellulin (BTC), epiregulin (EPR, EREG
– Mouse Genome Informatics) and epigen (EPGN). In mice that
lack specific ligands, such as TGFα, the absence of an HF
phenotype has been interpreted as functional redundancy among
ligands (Mann et al., 1993). By contrast, other work suggests that
active EGF signalling can inhibit HF development (Cohen and
Elliott, 1963). In skin organ culture, administration of EGF ligands
(EGF and TGFα) dramatically inhibits hair morphogenesis in E13.5
mouse skin (Kashiwagi et al., 1997). However, although these two
ligands are potent inhibitors of HF morphogenesis, they are not
endogenously expressed in the developing skin during follicular
morphogenesis. Our previous microarray analysis (Bazzi et al.,
2007a) indicated that amphiregulin is present specifically in the
epidermis, and its level of expression increases around the onset of
follicle initiation. This supports earlier reports of alternate EGFR
KGF and EGF signalling block hair follicle induction andpromote interfollicular epidermal fate in developing mouseskinGavin D. Richardson1, Hisham Bazzi2, Katherine A. Fantauzzo2, James M. Waters1, Heather Crawford1,Phil Hynd3, Angela M. Christiano2,4 and Colin A. B. Jahoda1,*
A key initial event in hair follicle morphogenesis is the localised thickening of the skin epithelium to form a placode, partitioningfuture hair follicle epithelium from interfollicular epidermis. Although many developmental signalling pathways are implicated infollicle morphogenesis, the role of epidermal growth factor (EGF) and keratinocyte growth factor (KGF, also known as FGF7)receptors are not defined. EGF receptor (EGFR) ligands have previously been shown to inhibit developing hair follicles; however, theunderlying mechanisms have not been characterised. Here we show that receptors for EGF and KGF undergo markeddownregulation in hair follicle placodes from multiple body sites, whereas the expression of endogenous ligands persistthroughout hair follicle initiation. Using embryonic skin organ culture, we show that when skin from the sites of primary pelageand whisker follicle development is exposed to increased levels of two ectopic EGFR ligands (HBEGF and amphiregulin) and theFGFR2(IIIb) receptor ligand KGF, follicle formation is inhibited in a time- and dose-dependent manner. We then used downstreammolecular markers and microarray profiling to provide evidence that, in response to KGF and EGF signalling, epidermaldifferentiation is promoted at the expense of hair follicle fate. We propose that hair follicle initiation in placodes requiresdownregulation of the two pathways in question, both of which are crucial for the ongoing development of the interfollicularepidermis. We have also uncovered a previously unrecognised role for KGF signalling in the formation of hair follicles in the mouse.
KEY WORDS: Hair follicle, Skin organ culture, EGF, KGF, Mouse
Development 136, 2153-2164 (2009) doi:10.1242/dev.031427
1School of Biological and Biomedical Sciences, University of Durham, DurhamDH1 3LE, UK. 2Department of Genetics and Development, Columbia University,New York, NY 10032, USA. 3School of Animal and Veterinary Science, The Universityof Adelaide, Roseworthy, South Australia 5371, Australia. 4Development Office,Columbia University, New York, NY 10032, USA.
*Author for correspondence (e-mail: colin.jahoda@durham.ac.uk)
Accepted 14 April 2009 DEVELO
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ligands, including HBEGF and amphiregulin, in skin at this time
(Kashiwagi et al., 1997), and suggests that EGFR ligands are
expressed dynamically in this developmental window.
Evidence that KGF signalling is functionally important for HF
development has come largely from mouse models, in which
knockout of the FGFR2(IIIb) receptor results in reduced HF density
and retarded follicle development (Petiot et al., 2003). Additionally,
overexpression of dominant negative FGFR2 also retards follicle
development (Werner et al., 1994), and removal of KGF ligand has
a modest effect on hair fibre texture (Guo et al., 1996). As with
EGF, reports on the effects of exposure of developing follicles to
excess ligand are stage-dependent. When recombinant KGF is
injected subcutaneously into adult athymic nude mice over 17 to 18
days, it induces dose-dependent hair growth (Danilenko et al.,
1995). However, overexpression of KGF in the basal epidermal and
outer root sheath (ORS) keratinocytes via the K14 promoter blocks
follicle development (Guo et al., 1993). One interpretation is that
the effects of KGF on HF morphogenesis are likely to be dependent
on the dose, time and site of KGF production in skin (Botchkareva
et al., 1999). A similar hypothesis has been suggested for EGF
signalling, whereby the fate of interfollicular versus follicular
epithelium at the onset of follicle morphogenesis is determined
partly by the levels of exogenous EGFR ligands (Kashiwagi et al.,
1997).
In this study, we revisited the role of EGFR and FGFR signalling
during normal HF morphogenesis. Initially, we defined the kinetics
of expression of the two receptors, both of which were found to be
universally downregulated in placodes of all follicle types examined.
We identified endogenous ligands associated with EGF and FGF
signalling in dermis and epidermis at the start of HF morphogenesis.
We found that constitutive activation of both EGF and KGF
signalling in the epidermis led to follicle inhibition in a process that
was dose dependent but that did not alter the patterning of residual
follicles. To probe the molecular events involved in this inhibition,
separated epidermis and dermis from treated organ-cultured skins
were analysed by whole genome microarray. The resulting data,
supported by in situ hybridisation and immunohistochemistry,
produced a molecular profile that confirmed the promotion of
interfollicular fate at the expense of HF development.
Pharmacological inhibition of EGF signalling alone had no
influence on normal skin development, highlighting the importance
of KGF signalling in these events. We propose that both EGF and
KGF receptors are downregulated as part of a mechanism that
requires abrogation of signalling via these pathways for placode
formation to occur.
MATERIALS AND METHODSSkinTo investigate in vivo skin, mystacial pad skin aged between E12.5 and
E17.5, dorsolateral skin between E13.5 and E17.5, and tail skin at E18.5
were microdissected from C57/Bl6J mice, embedded in TissueTek OCT
Compound (Agar Aids), snap frozen and stored at –80°C.
Organ cultureOrgan culture of back skin was performed using a procedure modified from
that described previously (Kashiwagi et al., 1997). To standardise
experiments, discrete and consistently sized pieces of dorsolateral skin (~1
� 2 mm2) were microdissected from the same region of C57/Bl6J mouse
embryos at E13.5 (n=198). Skin was then placed epidermal side up onto rat
tail collagen type 1 (Sigma) coated Nucleopore filters (pore size 8 μm;
Whatman) floating on 2 ml of DMEM containing 1% Pen-Strep, 1%
Fungizone and varying concentrations of EGF (Sigma), HBEGF (R&D
Systems), amphiregulin (R&D Systems), KGF/FGF7 (R&D Systems), the
EGF receptor inhibitor AG1478 (Calbiochem) or BSA control (Sigma) in a
35 mm dish. Skin samples were moistened with small amounts of media and
incubated in a humidified atmosphere containing 5% CO2 for 6, 24 or 72
hours.
Individual skin samples were imaged using a KY-F1030 digital camera
(JVC) fitted to a Stemi SVII dissecting microscope (Carl Zeiss) using both
bright- and dark-field settings to visualise external follicle structures. When
required, intact skin specimens were split with trypsin/pancreatin to facilitate
quantitation (below) of developing follicles.
Cultured specimens were embedded in TissueTek OCT Compound, snap
frozen and stored at –80°C or fixed in 4% paraformaldehyde in PBS
overnight at 4°C for in situ hybridisation or paraffin wax embedding. For
each experimental culture condition, at least six replicates were performed,
involving a total of 258 back skin samples and 54 mystacial pad specimens.
Follicle analysisImages of specimens were taken as above and analysed using UTHSCSA
ImageTool software (http://ddsdx.uthscsa.edu/dig/itdesc.html); the area was
calculated and total follicle numbers were counted. Individual follicles
within regions of highest visible follicle density were chosen, and linear
distances from the centre of the follicle to the centre of its nearest neighbours
were measured. Means were calculated from the results of multiple (>3)
measurements.
Immunofluorescence and immunohistochemistryCryosections (7 μm) were thaw-mounted on poly-L-lysine coated slides and
methanol acetone-fixed. The following antibodies were used for specific
labelling: anti-P-cadherin, 1:50 (clone P-CAD, ZYMED); anti-CD44-RPE
(clone KM201, Serotec); polyclonal anti-BEK (FGFR2; C17) and
polyclonal anti-EGFR (100S, SantaCruz, Autogen Bioclear UK, Wiltshire,
UK), polyclonal anti-CD138 (syndecan 1; 281-2, BD Pharmingen, Oxford,
UK), polyclonal EDAR (AF745, R&D Systems, Minneapolis, MN, USA),
polyclonal anti-LEF1 (CL2A5, Cell Signaling Technologies, Danvers,
USA).
Whole mount in situ hybridisationThe β-catenin probe was a kind gift from Dr Sarah Millar (University of
Pennsylvania, Philadelphia, Pennsylvania, USA). The Gli1 probe was a kind
gift from Dr Alexandra L. Joyner (Memorial Sloan-Kettering Cancer Center,
New York, NY, USA). Whole mount in situ hybridisation was performed on
cultured embryonic skin as per published protocols (Wilkinson, 1998).
Semi-quantitative RT-PCRTotal RNA was treated with DNaseI (Invitrogen). First strand cDNA was
prepared from 25 ng total RNA using 0.5 μg of Oligo(dT) (Invitrogen) and
200 U of SuperScript II RT (Invitrogen). Polymerase chain reactions (PCRs)
were performed as per manufacturer’s instructions in a Peltier Thermal
Cycler (MJ Research, Waltham, MA, USA). The different cDNAs from each
of the timepoints were equalised to β-actin. Aliquots were removed from the
total reaction after 25, 30 and 35 PCR cycles and electrophoresed on 1.0%
agarose/TBE gels containing 0.5 mg/ml of ethidium bromide, and imaged
using the Kodak Digital Science Electrophoresis and Documentation System
120.
Primers used for the analysis of endogenous expression of components
of the EGF and KGF signalling pathways in the dermis and epidermis
of dorsolateral skin (F, forward; R, reverse; 5′-3′): Areg-F,
CATCATCCTCGCAGCTATTG; Areg-R, TTGTCCTC AGCTAGG -
CAATG; β-actin-F, CCTGTATGCCTCTGGTCGTA; β-actin-R,
AAGGGTGTAAAACGCAGCTC; Egf-F, GATCCTATCACTG CAC -
ATGC; Egf-R, CAGTGCAAGTCTTCCCATCT; Egfr-F, GAGAGTG -
ACTGTCTGGTCTGC; Egfr-R, GATGGGGTTGTTGCTGAATC;
Fgfr2(IIIb)-F, GATGACCTTCAAGGACTTGG; Fgfr2(IIIb)-R, TTGTT -
GATA TCCCTGGCCAG; Hbegf-F, GGAAAGGGGTTAGGGAAGAA;
Hbegf-R, TCCTCTCCTGTGGTACCTAAACA; Kgf-F, AGGGTGAGA -
AGACTGTTCTG; Kgf-R, CTTTCCACCCCTTTGATTGC.
Primers used for the analysis of the expression of components of the EGF
and KGF signalling pathways in ligand-treated back skin: β-actin-F,
CCTGTATGCCTCTGGTCGTA; β-actin-R, AAGGGTGTA AAA -
CGCAGCTC; Egfr-F, GAGAACCTGCAGATCATCAG; Egfr-R,
ACCATGTTGCTTTGTTCTGC; Fgfr2(IIIb)-F, TCCTGGATCAGTGAG -
RESEARCH ARTICLE Development 136 (13)
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AATGTGGAG; Fgfr2(IIIb)-R, GCTTGGGGGCCCGTGAACACGC;
Hbegf-F, CCTTTTCAAAGTTGCTTTCTCC; Hbegf-R, TCCTCT -
CCTGTGGTACCTAAACA; Kgf-F, CAAACGGCTACGAGTGTGAA;
Kgf-R, TCCGCTGTGTGTCCATTTAG.
Microarray analysis of KGF and HBEGF-treated embryonic skinTriplicate culture experiments for each recombinant protein treatment were
performed as described above. Each experimental reiteration contained six
individual pieces of embryonic skin taken from littermates. This was
incubated as described above for 24 hours in HBEGF and KGF, washed in
Earle’s media and incubated in a mixture of 0.75% trypsin (without EDTA)
(Invitrogen) and filtered 2% pancreatin (Sigma-Aldrich, St Louis, MO,
USA) in Earle’s media for 20 minutes at 4°C. The epidermis was cleanly
separated from the dermis, and each tissue was dissociated in RLT buffer
(Qiagen, Valencia, CA, USA). Total RNA was isolated using the RNeasy
Mini Kit according to the manufacturer’s instructions (Qiagen). cDNA was
synthesised and labelled RNA samples were transcribed for hybridisation
on microarray chips (MOE430A) using Affymetrix reagents and protocols
(Affymetrix, Santa Clara, CA, USA). The data output was normalised and
analysed using GeneTraffic software (Iobion Informatics, La Jolla, CA,
USA). The BSA-treated control was set as a reference for comparison
purposes. The P-value cutoff was set to 0.05 and the significant fold
difference was considered twofold higher or lower than baseline.
RESULTSLocalisation of sites of EGFR and FGFR signallingduring hair follicle morphogenesisOur previous study of epidermal morphogenesis by global
transcriptional profiling (Bazzi et al., 2007a), revealed changes in
the expression of several genes that suggested that the EGF and
KGF pathways were active around the period of early follicle
morphogenesis. To determine the localisation of the EGF and KGF
receptor proteins, immunofluorescence was performed on
embryonic skin from different body sites. E13.5 back skin epidermis
consisted of a single layer of epithelial cells, which were all
expressing EGFR and FGFR2(IIIb) protein (Fig. 1A,G). At E14.5,
the epidermis was multiple layered and placodes had been initiated.
Intriguingly, EGFR and FGFR2(IIIb) expression was downregulated
to the extent that labelling was largely missing in placodes,
compared with the interfollicular epidermis (Fig. 1B,H). This
receptor downregulation occurs at a timepoint that corresponds with
activated Wnt signalling in the placode and DC, indicated by an
increased nuclear Lef1 expression in these early HF structures (Fig.
1H, insert). To ascertain whether loss of EGFR and FGFR2(IIIb)
was a common feature of all primary back skin follicles, or just a
subset, E15.0 skin was colabelled with the placodal marker P-
cadherin (cadherin 3 – Mouse Genome Informatics) and either
EGFR and FGFR2(IIIb). In all placodes, identified by P-cadherin
expression, both receptors were downregulated (n=593 from 3
samples; Fig. 1C,I). In E12.5 mystacial pad skin, the same absence
of EGFR and FGFR2(IIIb) protein was observed in the placodes
(Fig. 1D,J). This consistent pattern of diminished EGFR and
FGFR2(IIIb) expression was observed in the placodes of all follicle
types examined, including that of tail skin (Fig. 1E,K) and secondary
pelage follicles (Fig. 1F,M). The relative absence of both receptors
persisted in the epithelium during the hair germ and peg stages of
follicle development (see Fig. S1 in the supplementary material).
Splitting and recombining skin is one method of delaying and
synchronising follicle morphogenesis (Chuong et al., 1996). When
this was used to disrupt and delay follicle morphogenesis (see Fig.
S2 in the supplementary material) it did not ultimately affect
FGFR2(IIIb) receptor downregulation, highlighting that receptor
loss is linked specifically to placode formation, rather than a
chronological timepoint.
Analysis of endogenous expression of other components of the
EGF and KGF signalling pathways in the dermis and epidermis of
dorsolateral skin between E12.5 and E15.5 is shown in Fig. S3 in the
supplementary material. HBEGF and amphiregulin were
omnipresent in the epidermis. EGFR, the receptor for these ligands,
was identified at the transcript level in the developing epidermis at
all stages, with a sharp drop in expression at E14.5, coinciding with
HF initiation. Transcripts for KGF were only detected in the dermis.
KGF receptor (FGFR2) transcripts were present in both the
epidermis and dermis at all timepoints.
Activation of EGF and FGF signalling byendogenous ligands specifically blocks hairfollicle initiationThe focal loss of expression of EGFR and FGFR2(IIIb) within
placodes led us to investigate the effects of constitutively activated
EGF and KGF signalling on HF morphogenesis. After 72 hours,
developing follicles were visible in control skin cultures as rounded
2155RESEARCH ARTICLEControl of hair follicle morphogenesis by KGF and EGF
Fig. 1. EGFR and FGFR2(IIIb) expression is downregulated in hairfollicle placodes. (A-M) Mouse skin sections from back, whisker padand tail regions labelled with antibodies specific to EGFR (A-F), orFGFR2(IIIb) (G-M) (green). EGFR and FGFR2(IIIb) expression is uniform inback skin epidermis at E13.5 (A,G) and reduced in the placodes(arrows) of back skin follicles by E14.5 (B,H) and in all other developingfollicles (D-F,J-M). At E14.5 LEF1 is upregulated in the nucleus of cells inthe placode and early DC (H insert), costaining with placodal marker P-cadherin (red) and EGFR (green) (C) or P-cadherin (red) and FGFR2(IIIb)(green) (I). Insert shows receptor staining only. All images show nuclearDAPI (blue). Dotted lines mark the boundaries between epidermis anddermis. Scale bars: 60μm. ep, epidermal placode.
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external structures distributed across the whole skin surface,
including the edges (Fig. 2A). Haematoxylin and Eosin (H&E)
staining revealed well-developed hair germs and a multi-layered
epidermis starting to cornify (Fig. 2B). EGF, HBEGF and
amphiregulin were selected to test the effect of EGFR ligands on the
formation of HFs, and each was found to inhibit follicle formation,
although at different potencies: 50 ng/ml of recombinant EGF was
capable of completely blocking the presence of any discernable
follicular structures on the skin surface (Fig. 2C); and 250 ng/ml of
HBEGF was required to achieve the same effect (Fig. 2E).
Amphiregulin was the least potent, and at concentrations up to 500
ng/ml a small number of follicles were observed externally (Fig.
2G). In addition to follicle inhibition, all of the EGFR ligands
elicited numerous folds/wrinkles in the treated skins (Fig. 2C,E,G).
When examined histologically, skin cultured with either EGF (50
ng/ml) or HBEGF (250 ng/ml) had no HF structures in 39 out of 43
cultures (91%; Fig. 2D,F) and thicker, well-cornified epidermis. The
wrinkles/folds observed on the surface of the skin were clearly
visible as deep downward epidermal projections into the dermis
(Fig. 2D,F,H).
KGF (250 ng/ml) also completely inhibited follicle
morphogenesis, as seen externally and histologically. These
specimens had cornified epidermis that was uniformly thicker than
that of the controls but no folding or wrinkling (Fig. 2I,J). However,
another FGFR2(IIIb) ligand, FGF10, was unable to inhibit follicle
morphogenesis (Fig. 2K,L) at the same concentration (250 ng/ml).
To verify that structures visible externally were developing HFs,
cultured skin was enzymatically separated into epidermis and
dermis. In all cases, the number of surface bumps equalled the
number of obvious developing follicles in the epidermis after
separation (Fig. 2M). No follicles were observed in separated skin
that had been cultured with 250 ng/ml HBEGF (Fig. 2N) or KGF
(data not shown). EGF, HBEGF and KGF administered to E14.5
dorsolateral embryonic skin, in which EGFR- and FGFR2(IIIb)-
negative placodes had already formed, elicited no inhibitory effect
(see Fig. S4 in the supplementary material).
Inhibition of follicle morphogenesis by EGFR andFGFR2(IIIb) activation is dose dependent,morphology and patterning of residual hairfollicles is normalTo investigate the dose-dependent inhibition of HF formation, skin
cultures were established with increasing concentrations of each
recombinant ligand, and follicle density was calculated. Results for
HBEGF and FGF10 are shown in Fig. 3A. Control skin cultured for
72 hours in the absence of ligand developed 38±4.8 follicles per
millimetre squared; this density was uniformly distributed across the
skin surface. Upon treatment with increasing concentrations of
HBEGF, HF numbers were correspondingly reduced. With 50 ng/ml
they were halved to 19.5±7.9 (P<0.0005), they dropped to 7.5±5.4
(P<0.000013) with a dose of 125 ng/ml, and no follicles were visible
at the highest dose of 250 ng/ml (Fig. 3A). Having observed no
inhibitory effects using FGF10 at a concentration at which KGF was
inhibitory (250 ng/ml; Fig. 2I), we showed that specimens treated
with a range of concentrations of FGF10 up to 500 ng/ml were
indistinguishable from controls, and showed no significant
difference in follicle density (Fig. 3A).
Follicles that developed in the presence of lower dosages of
HBEGF, AR and KGF were morphologically indistinguishable
from those in the control skins and expressed normal placodal
markers, including EDAR (Fig. 3B). Intriguingly, on HBEGF-,
AR- and KGF-treated skin samples in which follicles had
developed, the follicles were not evenly distributed, but instead
were localised in clusters. For HBEGF, the distance between the
central follicle in each follicular cluster and its seven closest
RESEARCH ARTICLE Development 136 (13)
Fig. 2. Constitutive EGF and FGF signalling can inhibit hair folliclemorphogenesis. (A-L) E13.5 back skin organ-cultured with BSA (A,B)or a recombinant growth factor (C-L) for 72 hours (n=42 for eachtreatment). (A,C,E,G,I,K) Surface of the skin following culture. For skinstreated for 72 hours with BSA (250 ng/ml, A) HFs are visible on thesurface. Follicles could not be identified on EGF-(50 ng/ml, C), HBEGF-(250 ng/ml, E), or (I) KGF-(250 ng/ml) treated skins. (G) Amphiregulin(AR) was less potent, and blocked the majority of (but not all) folliclesat 500 ng/ml. (K) FGF10 (250 ng/ml) had no visible effect on HFdevelopment. (B,D,F,H,J,L) H&E staining of skin sections. Arrowsindicate: hair germs (yellow arrows); DCs (black arrows); epidermalfolding (blue arrows); epidermal ridges (white arrows). Follicle countswere verified by enzymatically splitting cultured skin and confirmingnormal follicle numbers in control epidermis (M) and follicle inhibitionin treated epidermis (N). Scale bars: 0.5 mm in A,C,E,G,I,K,M,N; 60μmin B,D,F,H,J,L.
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neighbours was quantified for each of the specimens (n=18) at 0
ng/ml, 50 ng/ml (P=0.0006) and 125 ng/ml (P=0.02) (Fig. 3C).
No significant increases in interfollicular spacing were found
between control and treated samples (Fig. 3D). The same analysis
of FGF10-treated samples revealed no increase in follicle spacing
between control and treated samples (Fig. 3D).
Absence of hair follicle placodes and dermalcondensates in EGF- and FGF-treated skinTo investigate at the molecular level the apparent absence of
follicles and whether epidermal folding might represent vestigial
follicle structures, we studied the expression of three follicle
markers: P-cadherin, which identifies placodes (Jamora et al.,
2003); and CD44 and syndecan 1, which both identify dermal
condensates (Hayashi et al., 2002; Underhill, 1993). The three
proteins were highly expressed in their appropriate HF structures
relative to interfollicular dermis and epidermis, both in control
cultured skin and equivalently staged in vivo skin (Fig. 4A-D,K-
P). Skin cultured with EGF, HBEGF or KGF for 24 or 72 hours
revealed an absence of strong differential HF-associated
expression of P-cadherin (Fig. 4E-J), CD44 (Fig. 4Q,T,W) and
syndecan 1 (Fig. 4R,S,U,V,X,Y). However, KGF treatment
resulted in a uniform layer of strong ectopic syndecan 1
expression beneath the basement membrane (Fig. 4V,Y). This is
possibly due to an FGF-inducible response element on the
syndecan 1 gene (Jaakkola et al., 1997).
Inhibition of vibrissa follicle development byactivated EGF and FGF signalling occurs in adefined spatiotemporal orderVibrissa follicles develop at around E12.5 in mice (Ibrahim and
Wright, 1975) in a precise pattern of defined rows and columns. The
latter are delineated by letter, starting with a (containing the largest and
most posterior follicles), and rows are delineated by number, starting
with 1 (the most dorsal row) (Oliver, 1966). During development,
follicles develop spatiotemporally in a posterior-to-anterior direction,
therefore column a and b follicles are the first to appear.
We investigated the effects of HBEGF and KGF ligand treatment
on whisker pads from faces of E13.0 embryonic mice (Fig. 5A-C),
at which point follicles in columns a, b, and some in column c, had
begun to develop. The majority of follicles in column c and all
follicles that would make up columns d through to f had yet to form,
and therefore these regions were still expressing both EGFR and
FGFR2(IIIb). Following 72 hours of culture, the control BSA-
treated (250 ng/ml) skin developed in a manner equivalent to the in
vivo process, displaying all rows a through f, as well as smaller
follicles that appear at the nasal (anterior) end of the face (Fig.
5D,D�). By contrast, skin samples treated with HBEGF or KGF
showed arrested follicle patterning. No additional follicles were
observed on the skin surface at the anterior end of the whisker pad
(Fig. 5E-F�), as confirmed by immunofluorescence analysis (Fig.
5G-I). Those posterior follicles that were present at the start of
culture, however, continued to develop normally (Fig. 5E,F,K,L,M).
2157RESEARCH ARTICLEControl of hair follicle morphogenesis by KGF and EGF
Fig. 3. HBEGF, but not FGF10, inhibitsfollicle morphogenesis in a dose-dependent manner but morphology andpatterning of residual hair follicles arenormal. (A) The number of follicles againsttreatment with increasing concentrations ofHBEGF or FGF10 after 72 hours in culture;only HBEGF treatment elicits a dose-dependent reduction in follicle numbers.(B) Skin treated with 125 ng/ml of HBEGFstained with H&E (left) or labelled with anti-EDAR antibody (green), nuclei stained withDAPI (blue) (right). Residual follicles thatescaped inhibition appeared morphologicallyindistinguishable from those in controlcultured skin. (C) Following treatment of skinwith up to 125 ng/ml of HBEGF, residualfollicles were present in islands or groups.(D) Graphs represent the distance between atleast four follicles from each skin sample (n=6)for each treatment concentration (of eitherHBEGF or FGF10) and their seven nearestneighbours within these groups (illustrated inC). Scale bars: 60μm in B; 0.5 mm in C.
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Downregulation of Wnt and SHH signalling in EGF-and FGF-treated skinTo interrogate the status of key signalling pathways active during
early pelage HF morphogenesis, we combined organ culture,
microdissection and microarray analysis. Skin taken from treated and
control skin cultures was enzymatically split into dermis and
epidermis and transcriptional profiling was performed as we described
previously (Bazzi et al., 2007a). After 24 hours of treatment with
either KGF or HBEGF, we noted a marked reduction in many of the
recognised molecular signatures of HF morphogenesis. These
included key effectors in the Wnt, (LEF1, DKK4), sonic hedgehog
(SHH, GLI1 and patched homolog 2) and BMP (BMP2) signalling
pathways (Table 1). In mice, Wnt signalling is required for the early
events of follicle initiation, whereas the SHH pathway is associated
with later stages of follicular morphogenesis. To verify the
downregulation of the Wnt and SHH pathways whole mount in situ
hybridisation was performed. In control skin after 24 hours of culture,
β-catenin mRNA had accumulated in developing follicles
corresponding to cells in which the Wnt signalling pathway is
activated (Fig. 6A). Gli1 mRNA transcript expression represents a
readout for activation of the SHH signalling pathway, and at 72 hours
Gli1 mRNA was concentrated in a ring pattern (Fig. 6A). Sectioning
of skin at both timepoints confirmed focal expression of Gli1 in the
dermis of the developing follicles (Fig. 6B). In EGF-, HBEGF- and
KGF-treated embryonic skin, no follicular structures and no discrete
accumulation of β-catenin beyond background levels was evident
(Fig. 6A,B). To investigate the early effects of EGFR or FGFR2(IIIb)
activation on Wnt signalling, we cultured day E13.5 skin with control
BSA, HBEGF or KGF for 6-hour and 24-hour timepoints and
evaluated the nuclear expression of LEF1, an established marker of
RESEARCH ARTICLE Development 136 (13)
Fig. 4. P-cadherin, CD44 and syndecan 1 confirm the absence of placodes and dermal condensations in ligand-treated skin. (A-Y) P-cadherin, CD44 and syndecan 1 expression (red) in ligand-treated mouse back skin. P-cadherin is upregulated at sites of placode formation duringHF morphogenesis (A,B). CD44 and syndecan 1 are expressed only in the DC within the dermal compartment of embryonic skin during HFmorphogenesis (K-M). Controls show a distribution of P-cadherin, CD44 and syndecan 1 comparable with in vivo embryonic skin (C,D,N-P). Skincultured with EGFR ligands EGF (50 ng/ml) or HBEGF (250 ng/ml) displayed no localised upregulated P-cadherin expression (E-H) and no differentialdermal expression of CD44 or syndecan 1 (Q-V). Skin cultured with KGF (250 ng/ml) displayed no localised upregulated P-cadherin expression (I,J)or CD44 (W). KGF treatment resulted in a continuous layer of syndecan 1 expression in the dermal cells subjacent to the epidermis (X,Y). P-cadherin, CD44 and syndecan 1 (red), laminin (green), nuclear DAPI (blue). Scale bars: 60μm.
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activated Wnt signalling. At 6 hours, control skin expressed nuclear
LEF1 in the epidermis and, at a low level, in some dermal cells.
Following 24 hours, cells in placodes and DCs were highly stained by
the LEF1 antibody, in a manner comparable to that observed in vivo
(Fig. 1H). Treatment with HBEGF or KGF resulted in an absence of
discrete LEF1 signalling in both compartments at either the 6-hour or
24-hour timepoints. Interestingly, treated skins continued to express
nuclear LEF-1 at all culture timepoints investigated but without a focal
distribution (Fig. 6C).
HBEGF and KGF treatment promote aninterfollicular epidermal phenotypeKGF and HBEGF treatment resulted in the rapid induction of genes
involved in epidermal terminal differentiation. These genes included
S100a18 (Hrnr – Mouse Genome Informatics), S100a6, loricrin,
keratin 6A, Tgm1 K (Tgm1), Lgals3, Lgals7 and filaggrin. Moreover,
S100a18 and keratin 6A, which are normally absent until E15.5 in
vivo (Bazzi et al., 2007a), were expressed in cultured skin equivalent
to E14.5 and therefore appeared prematurely (Table 2). To validate the
microarray analysis, immunofluorescence for keratin 6A, filaggrin
and loricrin was performed on treated cultures. The expression of each
was increased in the epidermis from treated skin compared with the
control (see Fig. S5A-I in the supplementary material).
Blocking of EGFR using AG1478 has no effect onhair follicle morphogenesis in organ-cultured skinTo investigate the effects of the inhibition of EGFR signalling on
HF morphogenesis, E13.5 skin was organ cultured with AG1478,
an inhibitor of EGF receptor kinase autophosphorylation.
AG1478 is specific for EGFR at concentrations up to 100 μM
2159RESEARCH ARTICLEControl of hair follicle morphogenesis by KGF and EGF
Fig. 5. Treatment of organ-cultured E13.0 whisker pads with HBEGF or KGF inhibits new vibrissa follicle initiation but does notinfluence the development of existing follicles. (A-F�) Whisker pads at E13.0 in which the two most posterior columns of follicles had alreadybegun to develop were cultured for 48 hours with BSA (250 ng/ml; control) (A,D,D�) or either HBEGF (250 ng/ml) (B,E,E�) or KGF (250 ng/ml)(C,F,F�). The control BSA-treated whisker pads (A,D,D�) continued to develop vibrissa follicles in the anterior direction ; (D′) yellow dots used tohighlight the presence of follicles. Whisker pads treated with HBEGF (B,E,E�) or KGF (C,F,F�) failed to develop any ‘new’ follicles during the 48 hoursof culture. (G-L) Whisker pads were sectioned and labelled with antibodies specific to syndecan 1 (red) and laminin (green). Analysis of the folliclesin the posterior region showed that HBEGF (K) and KGF (L) had no effect on the development of existing HFs or the formation of the DP. Analysis ofHBEGF-(H) or KGF-(I) treated skin in the anterior region of the whisker pads revealed no epidermal HF structures or DCs. Scale bars: 60μm. dc,dermal condensation; dp, dermal papilla; hf, hair follicle.
Table 1. Genes implicated in epidermal differentiation that are downregulated in the epidermis following 24-hour culture withEGF or KGF ligandGene Control KGF HBEGF
sonic hedgehog 0 –9 –9dickkopf homolog 4 (Xenopus laevis) 0 –9 –13GLI-Kruppel family member GLI1 0 –8 –10periostin, osteoblast specific factor 0 –5 0patched homolog 2 0 –5 –12bone morphogenetic protein 2 0 –3 –12cut-like 1 0 –3 –3lymphoid enhancer binding factor 1 0 –2 –2
Embryonic skin was cultured with BSA (control) (250 ng/ml), HBEGF (250 ng/ml) or KGF (250 ng/ml) for 24 hours and genomic transcript expression was profiled bymicroarray. A number of genes linked to major pathways (Wnt, sonic hedgehog, BMP) involved in HF morphogenesis are downregulated following HBEGF or KGF treatment.0, baseline levels or no change; numbers, fold of baseline. D
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2160
(Gazit et al., 1991), and was previously shown to cause fusion of
interbud rows at a concentration of 50 μM (Atit et al., 2003).
Following 72 hours of culture with AG1478 at a concentration of
100 μM, treated skin specimens (n=12) appeared
indistinguishable from controls. Skin was split (n=3) to reveal
individual hair pegs with no visible fusion (data not shown).
Histology and immunofluorescence staining with anti-syndecan
showed normal follicles comparable with controls (Fig. 7A,B).
Finally, measurement showed that there was no significant
difference in follicle number or interfollicular spacing between
treated and control skin specimens (Fig. 7D,E).
Interplay between EGF and KGF signalling inligand-treated organ-cultured skinTo investigate how short-term treatment with HBEGF or KGF
influenced the expression of each other, and their own receptors,
E13.5 mouse back skin was cultured in the presence of BSA (250
ng/ml), HBEGF (250 ng/ml) or KGF (250 ng/ml) for 6 hours.
Dermis and epidermis were then enzymatically separated and the
expression of Hbegf, Egfr, Kgf and Fgfr2(IIIb) mRNA transcripts
was investigated using semi-quantitative RT-PCR. HBEGF
treatment appeared to have no effect on the expression of either
the ligands (Hbegf and Kgf) or the receptors [Egfr and
Fgfr2(IIIb)] of either pathway when compared with controls (Fig.
8). However, KGF treatment upregulated Kgf expression in the
dermis, and in the epidermis it produced increased Hbegf and
decreased Fgfr2(IIIb) expression, but no change in Egfrexpression.
DISCUSSIONEGF and KGF signalling pathways are conventionally seen as
promoting cell growth and proliferation in the interfollicular
epidermis (Beer et al., 2000; du Cros, 1993; Peus and Pittelkow,
1996; Schneider et al., 2008). However, mouse models with
elements of these pathways disrupted in skin have indicated that
activated EGF and KGF signalling are both crucial for HF
morphogenesis and development (Danilenko et al., 1995; Guo et al.,
1996; Miettinen et al., 1995; Murillas et al., 1995; Petiot et al., 2003;
RESEARCH ARTICLE Development 136 (13)
Fig. 6. Wnt and SHH pathways fail to activatein HBEGF- and KGF-treated skin. (A) Wholemounts of cultured embryonic skin. β-catenin andGli1 transcripts are expressed focally in the controlcultures at 24 and 72 hours, respectively. HBEGF-,EGF- or KGF-treated skin samples, which have noexternal follicle structures, fail to show localisedexpression of β-catenin and Gli1 transcripts.(B) Sectioned whole-mount skin at 24 hours and 72hours showing upregulated Gli1 expression in thedermal condensation of BSA-treated controls, butnot in HBEGF- or KGF-treated samples. (C) E13.5back skin was cultured with either BSA or arecombinant growth factor for 6 (top) or 24(bottom) hours (n=4 for each treatment) andlabelled with antibody specific to LEF1 (green). At6 hours, there is no foci of LEF1 expression in eitherthe epidermis or dermis of all samples. At 24 hours,LEF1 expression was unregulated in a localisedmanner in the epidermal placode and dermalcondensation of BSA-treated controls, but not in theHBEGF- or KGF-treated samples. Scale bars: 60μm.
Table 2. Genes implicated in epidermal differentiation that areupregulated in the epidermis following 24-hour culture withEGF or KGF ligandGene Control KGF HBEGF
filaggrin 0 98 29involucrin 0 38 24loricrin 0 2 2keratin complex 2, basic, gene 6a 0 3 5keratin complex 2, basic, gene 6b 0 20 85keratin complex, acidic, gene 16 0 64 70S100a18 0 6 5S100a6 0 9 6transglutaminase 1, K polypeptide 0 2 2lectin, galactose binding, soluble 7 0 3 2small proline-rich protein 1A 0 10 6small proline-rich protein 1B 0 5 5small proline-rich protein 2A 0 12 18small proline-rich protein 3 0 144 25
Embryonic skin was cultured with BSA (control) (250 ng/ml), HBEGF (250 ng/ml) orKGF (250 ng/ml) for 24 hours and genomic transcript expression was profiled bymicroarray. Multiple genes involved in normal interfollicular keratinocytedifferentiation and barrier formation are upregulated following HBEGF or KGFtreatment. 0, baseline levels or no change; numbers, fold of baseline. D
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Werner et al., 1994). Here we demonstrate for the first time that
FGFR2(IIIb), as well as EGFR, are coordinately downregulated in
placodes of all mouse follicles. In a series of functional organ culture
experiments, we show that when skin from the sites of primary
pelage and whisker follicle development is exposed to increased
levels of two endogenous EGFR ligands (HBEGF and
amphiregulin) and the FGFR2(IIIb) receptor ligand KGF, follicle
formation is inhibited in a dose- and time-dependent manner. These
data demonstrate that active EGF and KGF signalling in the
placodes is prohibitive of HF formation. Since alterations in the
dermal condensate could alter placode formation, the question arises
as to whether the effects of the EGF and KGF ligands are due to
activation of signalling in the epidermis or the dermis? Our analysis
of placode and dermal condensate markers in treated skin at short
timepoints have revealed that condensates can briefly persist in the
dermis, but placodes are absent from the epidermis (see Fig. S6 in
the supplementary material). This, together with the exclusive
epidermal expression of the receptors, leads us to suggest that the
inhibitory effects of EGF and KGF signalling act initially on the
epidermis. Microarray profiling, and analysis of Wnt and SHH
pathway expression in treated skin, supports a model in which
failure to downregulate EGFR and FGFR2(IIIb) in HF placodes
leads to adoption of an interfollicular epidermal fate.
EGFR and FGFR2(IIIb) are coordinatelydownregulated during placode initiationPrevious reports have shown that the epidermal growth factor
receptor is downregulated in the basal cells of primary human
follicle placodes (Nanney et al., 1990). However, the concomitant
downregulation of FGFR2(IIIb) receptor specifically within the
epithelial placodes of developing primary follicles is a novel
observation that differs from previous reports (Danilenko et al.,
1995). We showed the same kinetics of expression in secondary
follicles, whisker follicles and tail skin follicles. Our results showed
that synchronised downregulation of EGFR and FGFR2(IIIb) appear
to be a universal process in follicle development, unlike for example
the tabby/downless/crinkled (EDA/EDAR/EDARADD) pathway,
which is associated with the development of a specific subset of
follicles (Mou et al., 2006). In order for one or both pathways to be
active, a receptor/ligand pair must be present in the tissue. Our
finding that expression of the EGF ligands HBEGF and
amphiregulin in the epidermis, and of KGF in the dermis, was
coincident with pelage follicle formation, is in general agreement
with previous observations on developing mouse (Kashiwagi et al.,
1997), rat (Dang et al., 2003) and human (Piepkorn et al., 1995) skin.
Inhibition of placodes by EGFR and FGFR2(IIIb)ligands: effects of exposure to ligands are dose-and time-dependent but do not perturbmorphogenesis and pattern formationKashiwangi et al. (Kashiwangi et al., 1997) demonstrated that two
EGFR ligands, EGF and TGFα, inhibited follicle development in a
dose-dependent manner in embryo skin organ culture. These authors
postulated that two other ligands, HBEGF and amphiregulin, could be
endogenous mediators of HF and epidermal development. Our study
showed that treating embryonic mouse skin with recombinant
HBEGF, amphiregulin or the FGFR2(IIIb) ligand, KGF, prior to
follicle initiation inhibited the development of follicular structures
dose dependently. One interpretation of the pronounced epidermal
folds observed in the EGF ligand-treated samples is that they were
aberrant or vestigial follicle structures. However, the absence of
placode (P-cadherin) (Jamora et al., 2003) and condensation
(syndecan 1 and CD44) (Trautman et al., 1991) markers showed that
this was not the case. Our findings correlate to some degree with
mouse models, whereby transgenic mice expressing the human
amphiregulin gene displayed a distinctive loss of follicles in the most
2161RESEARCH ARTICLEControl of hair follicle morphogenesis by KGF and EGF
Fig. 7. Inhibition of EGFR signalling has no effect on hair follicledevelopment or density. (A-E) Skins from E13.5 mice were culturedwith either BSA (250 ng/ml) or the EGFR inhibitor AG1478 (100μM).AG1478-treated skin surface (A). Antibody labelling for syndecan 1(red) and laminin (green), with nuclei labelled with DAPI (blue) (B) andH&E (C) revealed that follicles were indistinguishable from those of thecontrol (see Figs 2, 4). Follicular density and the average distancebetween follicles and their nearest neighbours were quantified (D,E).No significant difference between treated and control specimens wasobserved for either parameter. Scale bars: 60μm.
Fig. 8. The effects of HBEGF and KGF treatment on mRNAtranscript expression of ligands and receptors of EGF and KGFpathways in organ-cultured E13.5 mouse back skin. Semi-quantitative RT-PCR showing Hbegf, Egfr, Kgf and Fgfr2(IIIb) mRNAtranscript expression in the appropriate epidermal or dermalcompartment of E13.5 mouse back skin following 6 hours culture withBSA (250 ng/ml), KGF (250 ng/ml) or HBEGF (250 ng/ml). Comparedwith control specimens, skin cultured with HBEGF showed nodifference in transcript levels of epidermal expression of Hbegf, thedermal expression of Kgf, the epidermal expression of Egfr or theepidermal expression of Fgfr2(IIIb). Skin cultured with KGF for 6 hoursshowed an increased epidermal expression of Hbegf, an increase in Kgfdermal expression, and a decrease in epidermal Fgfr2(IIIb) transcriptexpression. KGF treatment does not alter epidermal expression of Egfr.
DEVELO
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2162
severely affected regions (Cook et al., 1997). Targeting of KGF
overexpression in mouse epidermis also resulted in a marked
suppression of HF morphogenesis (Guo et al., 1993). Intriguingly, we
showed that treatment with FGF10, an alternate ligand that binds to
the FGFR2(IIIb) receptor, produced no observable effect on follicle
development. Mice lacking FGF10 show a whisker follicle (Ohuchi
et al., 2003) but not a body HF (Suzuki et al., 2000) phenotype.
In agreement with previous studies using EGF (Kashiwagi et al.,
1997), we showed that when follicles from HBEGF-, amphiregulin-
and KGF-treated skin did escape inhibition (in the presence of lower
concentrations of ligand) they developed normally and expressed
normal HF-related markers. The fact that these residual follicles were
clustered with normal density suggests that there is localised failure
of follicular initiation in any given area, rather than a generalised
expansion of interfollicular epidermis increasing the distance between
follicles. Interestingly, some K14-hKGF (human KGF) transgenic
mice showed a mosaic pattern of skin and hair development, with
regions containing normal HFs alternating with areas possessing
thicker epidermis devoid of HF structures (Guo et al., 1993).
Previous work reported that after the start of follicle initiation,
skin is unresponsive to the inhibitory effects of EGF treatment
(Kashiwagi et al., 1997). Here, we showed that the same was true of
skin treated with the endogenously expressed ligands HBEGF and
KGF (see Fig. S4 in the supplementary material), emphasising that
follicle initiation is crucial in relation to EGF and KGF pathway
signalling and follicle morphogenesis. With vibrissa follicles on
whisker pads we were able to simultaneously show three different
results (loss of receptors in placodes, inhibition of HF induction with
excess ligand and continuation of normal development and
patterning in committed follicles), reinforcing both the findings with
primary pelage follicles and the universal nature of these signalling
events.
HBEGF and KGF promote epidermal fate at theexpense of HF developmentWe performed global transcriptional profiling to identify gene
pathways that are activated or inhibited in the epidermis after EGFR
and FGFR2(IIIb) signalling. Given the well-established role of
Wnt/β-catenin signalling for the initiation of HF development (Andl
et al., 2002; Zhang et al., 2008) it was noteworthy that Lef1, which
is a downstream effector of Wnt signalling, and the Wnt regulator
Dkk4 (Bazzi et al., 2007b), were both downregulated. Sonic
hedgehog, Gli1 and patched 2, key components of the SHH
signalling pathway involved later in follicular development (Chiang
et al., 1999; Karlsson et al., 1999; St-Jacques et al., 1998), were also
downregulated. These observations were verified with key
downstream mediators of the Wnt and SHH pathways, respectively,
using whole mount in situ hybridisation and antibody staining. This
showed an absence of distinct focal HF-related expression. They
demonstrate at a molecular level the complete failure of follicular
development following exposure to EGF and KGF signalling at the
pre-placode stage. Although there is a failure of placodal Wnt
signalling when E13.5 skin is cultured with ligands, we are not
suggesting that the activation of either receptor directly inhibits the
Wnt signalling pathway. Interestingly, as the epidermis of the treated
skins expressed nuclear LEF1 at all culture timepoints investigated,
albeit at low levels (Fig. 6C), it is attractive to suggest that EGFR
and FGFR2(IIIb) signalling does not inhibit the activation of the
Wnt signalling pathway.
Contrasting with this was the strong expression of genes
associated with epidermal differentiation, including S100 proteins
and small proline-rich proteins. Filaggrin, which is expressed
relatively late on in the stratification process (Bickenbach et al.,
1995), emerged prematurely in response to stimulation by both
ligands. Likewise, keratins 6 and 16, known to be involved in
epidermal homeostasis, as well as being expressed during the
‘activation’ of keratinocytes in wound healing, were also
upregulated. Taken together, these findings suggest that exposure to
EGF ligands and KGF promotes an interfollicular epidermal fate, at
the expense of HF morphogenesis (Guo et al., 1993; Schneider et al.,
2008).
The FGF pathway acts synergistically on the EGFpathway but not vice versaOur examination of the short-term influence of HBEGF and KGF
ligands on both pathways demonstrated that KGF upregulated its
own mRNA levels in E13.5 dermis, and that this was coupled with
decreased transcript levels of its receptor in the epidermis.
Interestingly, KGF treatment also strongly increased mRNA levels
of multiple EGFR ligands, including Hbegf, suggesting that KGF
can directly influence the EGF pathway. It has been shown
previously that KGF treatment of keratinocytes induces the
production of secreted TGFα protein, and that this is linked to the
downregulation of the EGF receptor (Dlugosz et al., 1994). The
reason we observed no EGF receptor downregulation could be a
matter of timing, since the above study was performed over days
compared with 6 hours here.
By contrast, HBEGF treatment appeared to have no effect on the
expression of either the ligands (HBEGF and KGF) or the receptors
[EGFR and FGFR2(IIIb)] of either pathway when compared with
control specimens (Fig. 8). However, auto- and cross-induction, and
autocrine signalling, occurs extensively among the EGF receptor
ligands (Barnard et al., 1994; Piepkorn et al., 1998) with HBEGF in
particular having a proposed juxtacrine interaction with its receptor
(Harris et al., 2003), which might be pertinent to placode formation
and the creation of a follicular/non-follicular cell boundary in
adjacent basal epidermal cells. Crucially, some EGF ligands are able
to downregulate the EGFR receptor (Singh and Harris, 2005) and,
more generally, different ligands are known to alter the magnitude
and duration of EGFR signals (Wells, 1999).
Denticles to feathers to hairs: alternating regionsof EGFR signalling establish regional specificitythroughout evolutionTreatment of embryonic chick skin with exogenous EGF suppresses
feather formation (Atit et al., 2003) in a manner similar to that seen
here with other EGF ligands and mouse HFs. Chick skin explant
cultures treated with AG1478, a recognised inhibitor of EGFR
signalling (Zieske et al., 2000) causes fusion of feather buds,
implying loss of interfollicular epidermal fate. In our study,
treatment of mouse skin with AG1478 at varying concentrations
failed to elicit any effect on development and patterning of pelage
and vibrissa follicles (data not shown). Similarly, in mice with
targeted disruption of EGFR, follicle morphogenesis is not disrupted
(Hansen et al., 1997). Thus, although in embryonic chick skin the
EGFR pathway might be sufficient to control interfollicular fate,
aspects of our work support the model that in mouse it does so in
conjunction with KGF signalling.
Why then, given the other well-established pathways that are
known to be involved in HF placode formation, do EGF and FGF
signalling downregulate locally in the placode, and what interactions
occur with other pathways? Concerning the first question, a trivial
answer is that it is a neutral response to a switch of fate involving
other mechanisms. Indeed, evidence from the literature suggests that
RESEARCH ARTICLE Development 136 (13)
DEVELO
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EDAR, for example, does not directly interact with EGF during
follicle initiation. A recent study showed that although both BMP4
and EGF are able to block the rescue of follicle formation by EDA
in Eda–/– mouse skin, BMPs but not EGF are able to repress Edarexpression in embryonic mouse skin prior to follicle patterning
(Mou et al., 2006). Among TGFβ family members, the TGFβ2
isoform appears to be particularly important for follicle
morphogenesis, as Tgfb2 null mice have reduced hair follicle
numbers, and adding TGFβ2 to developing skin in organ cultures
promotes epidermal hyperplasia and follicle induction (Foitzik et al.,
1999). Since all TGFβ isoforms have inhibitory effects on
keratinocyte cell proliferation in vitro, Foitzik et al. postulate that
the TGFβ2-specific influence on follicle development in vivo may
be indirectly influencing other growth factor pathways via different
cell types. Certainly, in vitro evidence militates against the idea that
TGFβ2 directly stimulates the KGF and EGF pathways; for
example, TGFβ2 dose-dependently inhibits corneal epithelial cell
proliferation promoted by KGF and EGF (Honma et al., 1997).
Likewise, in relation to early follicle development there are no
reports of direct interaction between noggin, an important antagonist
of BMP activity located in the mesenchyme (Botchkarev et al.,
1999) and KGF and EGF signalling. However, it is interesting that
the Notch pathway is linked to early follicle development (Favier et
al., 2000) and that EGFR has been recently identified as a key
negative regulator of notch1 gene expression in primary human
keratinocytes (Kolev et al., 2008). It is also noteworthy that AP2α ,
which is a negative regulator of the EGF receptor (Wang et al.,
2006), also becomes strongly expressed in the emerging HF placode
(Panteleyev et al., 2003).
Guo et al. (Guo et al., 1993) suggest that ‘elevated growth
response might block the mesenchymal-epithelial signalling
necessary for HF morphogenesis’. Certainly, one of the features
associated with placode formation is a reduction in epidermal cell
division (Mustonen et al., 2004; Wessells and Roessner,
1965).Therefore there may be a requirement to interrupt cell
division, to permit the transition from interfollicular to follicular
status and to create a new stem cell pool.
The Fuchs group has elegantly shown some of the cellular changes
that occur during hair placode initiation (Jamora et al., 2003). These
include a switch in expression from E-cadherin (cadherin 1 – Mouse
Genome Informatics), an adhesion molecule closely linked with the
EGFR pathway (Hazan and Norton, 1998), to P-cadherin, coincident
with other cytoskeletal and behavioural changes to the cells.
Therefore, the temporary downregulation of EGF and FGF signalling
may be integral to global changes to cell behaviour and directional cell
movements that occur in the placode.
Alternating regions of heightened and diminished EGFR
signalling is an evolutionarily conserved mechanism, for example,
it establishes vein patterning in Drosophila morphogenesis (Blair,
2007). Alternating regions of Wnt and EGFR signalling are also
hallmarks of patterning the denticles (Payre et al., 1999). These
mechanisms are reprised in the chick, where EGF signalling
specifies the interbud fate (Atit et al., 2003). That similar
mechanisms have been adapted for the formation of mammalian
HFs is perhaps not unexpected for EGF signalling. Now we have
uncovered a crucial overlapping role for KGF signalling in mouse
HF initiation, adding a new layer of complexity to an already
exquisitely regulated developmental process.
We thank members of the LSSU at Durham for their assistance. At ColumbiaUniversity we thank Mr Ming Zhang for excellent technical assistance and DrsVladan Miljkovic and Yonghui Zhang for expert assistance in microarrayanalysis. This work was partly supported by a generous grant from the Steven
and Michelle Kirsch Foundation (to A.M.C. and C.A.B.J.), by the New YorkState Foundation for Science Technology and Innovation (NYSTAR, to A.M.C.)and by the BBSRC (to C.A.B.J.). Deposited in PMC for release after 6 months.
Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/136/13/2153/DC1
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